The febrile response to lipopolysaccharide is blocked in cyclooxygenase-2^−/−, but not in cyclooxygenase-1^−/− mice

Abstract

Various lines of evidence have implicated inducible cyclooxygenase-2 (COX-2) in fever production. Thus, its expression is selectively enhanced in brain after peripheral exogenous (e.g., lipopolysaccharide [LPS]) or endogenous (e.g., interleukin-1) pyrogen administration, while selective COX-2 inhibitors suppress the fever induced by these pyrogens. In this study, we assessed the febrile response to LPS of congenitally constitutive COX-1 (COX-1^−/−) and COX-2 (COX-2^−/−)-deficient C57BL/6J-derived mice. COX-1^+/− and COX-2^+/− mice were also evaluated; controls were wild-type C57BL/6J mice (Jackson Labs.). All the animals were pretrained daily for two weeks to the experimental procedures. LPS was injected intraperitoneally at 1 μg/mouse; pyrogen-free saline (PFS) was the vehicle and control solution. Core temperatures (T_cs) were recorded using thermocouples inserted 2 cm into the colon. The presence of the COX isoforms was determined in cerebral blood vessels immunocytochemically after the experiments, without knowledge of the functional results. The data showed that the wild-type, COX-1^+/−, and COX-1^−/− mice all responded to LPS with a 1°C rise in T_c within 1 h; the fever gradually abated over the next 4 h. By contrast, COX-2^+/− and COX-2^−/− mice displayed no T_c rise after LPS. PFS did not affect the T_c of any animal. It would appear therefore that COX-2 is necessary for LPS-induced fever production.

Introduction

Much evidence has accumulated indicating that prostaglandin E₂ (PGE₂) is the proximal mediator of fever (reviewed in Refs. 1, 13). It is believed to be produced and released in consequence of an action of pyrogenic cytokines (endogenous pyrogens, EnP) which are themselves produced and released in response to invading infectious pathogens or to their products, exogenous pyrogens (ExP). PGE₂ is implicated as a fever mediator because, briefly: (1) it is a potent hyperthermic agent 1, 13thought to act directly or indirectly on thermoregulatory neurons in the preoptic anterior hypothalamus (POA), the primary brain site in which body temperature is regulated [2]; (2) its level increases and decreases in this brain region in conjunction with the febrile course 14, 43; (3) COX inhibitors, e.g., indomethacin and related nonsteroidal anti-inflammatory drugs (NSAIDs), inhibit pyrogen fever, in parallel with the reversal of PGE₂ synthesis [30]; and (4) the congenital absence of the PGE₂ EP₃ receptor impairs the febrile response to both ExP and EnP [47]. Although PGE₂ rises in blood promptly after the entry of microorganisms or after systemic ExP or EnP administration 31, 39, it is now generally agreed that the PGE₂ detected in the brain is not derived from the blood, but rather is produced directly in the brain 15, 33, 41, albeit that some species differences may exist [17]. However, the cell source and nature of the triggering mechanism that induces PGE₂ in the brain in response to systemic pyrogens, as well as its precise mode of action, remain controversial.

PGE₂ is formed by the cleavage of membrane phospholipids by phospholipases, yielding arachidonic acid (AA). The released AA, in turn, is converted into the prostaglandin endoperoxides, PGG₂ and PGH₂, via sequential cyclisation and oxygenation by cyclooxygenase (COX) and peroxidation by hydroperoxidase; these two enzymatic activities co-exist in a single protein, prostaglandin H synthase [44]. PGH₂ is then quickly isomerized to PGE₂ by PGE₂ isomerase. It is now recognized that there are at least two isoforms of COX which may [34]or may not [45]differ in their tissue and intracellular distributions, but are activated by distinct mechanisms. Thus, COX exists as a constitutive, COX-1, and an inducible, COX-2, isoenzyme; each is encoded by separate genes [35], but the enzymes share 60 to 70% homology 24, 50. COX-2 is up-regulated by proinflammatory mediators in, among other cell types, stimulated, but not unstimulated, macrophages and endothelial cells; it is selectively down-regulated by anti-inflammatory glucocorticoids. COX-2 is, however, also expressed constitutively in unstimulated neurons 4, 5, 6, 20, 25, 36, 37, 49, but the data differ on whether it is also up-regulated by pyrogenic stimuli 3, 11, 16, 18. By contrast, COX-1 is constitutively expressed in most cells; it is not affected by inflammatory mediators, and its basal activity is not altered by glucocorticoids (reviewed in Refs. 22, 23, 46).

Because of its inducibility by inflammatory mediators, it may be anticipated that COX-2 should have a major role in the brain in fever production. Indeed, it is now well-documented that ExP (e.g., bacterial endotoxic lipopolysaccharides [LPS]) and EnP (e.g., interleukin-1β [IL-1β]) activate COX-2 in vivo. Thus, in recent studies, COX-2-like immunoreactivity [28]and COX-2 mRNA 10, 11, 18, 37were found to be expressed in rat cerebral microvascular endothelial cells, particularly venules, ca. 1.5 h after intraperitoneal (i.p.) LPS and in perivascular microglia and meningeal macrophages ca. 2.5 h after intravenous (i.v.) LPS administration. In contrast, COX-1 expression was not affected anywhere in the brain by the peripheral administration of pyrogens. Moreover, treatment with specific inhibitors of COX-2 (NS-398, L-745, 337, DFU) orally after i.v. LPS [21]or intraperitoneally before i.p. LPS 11, 12, 38suppressed the febrile response, but did not affect basal body temperature. These antipyretic effects were not different from those produced by conventional NSAIDs, which inhibit both COX-1 and COX-2. Taken together, therefore, these data would seem to provide compelling support for the critical importance of COX-2 in fever genesis. To substantiate this inference, we examined the febrile response to LPS administered intraperitoneally of COX-1 and COX-2 gene knockout mice.